Lyophilized platelet lysates

ABSTRACT

The present disclosure is drawn to compositions and methods of making and using lyophilized platelet lysates. Specifically, a method of preparing a composition suitable for therapeutic use or as a culture medium can comprise steps of concentrating platelets from a platelet source to form a platelet rich portion of the platelet source, and lysing the platelets in the platelet rich portion to form a plurality of lysates. An additional step includes lyophilizing the lysates to form lyophilized platelet lysates in a composition with released concentrations of available growth factors, cytokines, and chemokines. In one example, at 30%, by platelet count, of platelets from a platelet source can be lysed using this process.

The present application is a divisional application of U.S. patentapplication Ser. No. 13/358,797, filed on Jan. 26, 2012, which isincorporated herein by reference.

BACKGROUND

Several techniques have been described for the preservation and use ofplatelets. DMSO and trehalose are two examples of compositions that havebeen used for the perseveration of platelets, with or withoutlyophilization (or freeze-drying). Cryoprotectant compositions have alsobeen used along with lyophilization processes to a similar result.Disadvantages with approaches that merely use preservatives and/orlyophilization on intact platelets relate to the fact that theseplatelets retain their proteins, receptors, and the like on the surfaceor within the platelets. For example, several platelet membranereceptors remain intact for binding with extracellular factors inresponse to platelet activation, e.g., for platelet adhesion,aggregation, etc. Thus, it would be beneficial to prepare a moreuniversal preparation of platelets that can be used effectively for manyapplications, including wound healing, skin treatment, disorders of bodytissue (including body organs) such as lung tissue, or the like.

SUMMARY

A method of preparing a composition suitable for therapeutic use or as aculture medium can comprise steps of concentrating platelets from aplatelet source to form a platelet rich portion of the platelet source,and lysing the platelets in the platelet rich portion to form aplurality of lysates. An additional step can include lyophilizing thelysates to form lyophilized platelet lysates in a composition withreleased concentrations of available growth factors, cytokines, andchemokines. In one example, at least 30%, by platelet count, ofplatelets from a platelet source can be lysed (during the lysing stepand often the lyophilizing step as well) using this process.

In another example, a composition suitable for therapeutic use or as aculture medium can comprise lyophilized platelet lysates prepared fromsource platelets, wherein the lyophilized platelet lysates providereleased concentrations 1011 of available growth factor, cytokines, andchemokines. In one example, at least 30% of the source platelets thatremain in the composition, by platelet count, are lysed to form thelyophilized platelet lysates.

In another embodiment, a method of treating mammalian tissue cancomprise applying a composition including lyophilized platelet lysatesto a mammalian tissue site for treatment, wherein the lyophilizedplatelet lysates include released concentrations of available growthfactors, cytokines, and chemokines. In one example, the lyophilizedplatelet lysates are prepared from source platelets, wherein at least30% of the source platelets that remain in the composition, by plateletcount, are lysed to form the lyophilized platelet lysates.

A method of culturing cells or tissue can comprise admixing a mediacomposition including lyophilized platelet lysates with a cell or tissueculture. The media composition can include lyophilized non-lysedplatelets as well, and at least 30% of a total platelet count can makeup lyophilized platelet lysates.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 sets forth a comparison of mesenchymal stem cell cultures usingvarious types of media, one of which includes lyophilized plateletlysates prepared in accordance with examples of the present disclosure.

DETAILED DESCRIPTION

Reference will now be made to the exemplary embodiments and specificlanguage will be used herein to describe the same. It will neverthelessbe understood that no limitation of the scope of the invention isthereby intended. Alterations and further modifications of the inventivefeatures illustrated herein, and additional applications of theprinciples of the disclosure as illustrated herein, which would occur toone skilled in the relevant art and having possession of thisdisclosure, are to be considered within the scope of the invention. Itis also to be understood that this disclosure is not limited to theparticular configurations, process steps and materials disclosed herein,as these may vary to some degree. Further, it is to be understood thatthe terminology used herein is used for the purpose of describingparticular embodiments only, and is not intended to be limiting as thescope of the present disclosure.

It is noted that, as used in this specification and the appended claims,singular forms of “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise.

The term “wound” refers to any damage to any tissue of a subject,including damage to the skin as well as damage to deeper tissue, whetherthe wound is caused accidentally or intentionally, or alternatively bythe normal course of a pathological, disease, or degenerative condition.For example, the damage can be as a result of injury or surgery.Non-limiting examples of injuries include ulcers, burns, broken bones,punctures, cuts and scrapes, lacerations, surgical incisions,inflammation, infection, and the like.

As used herein, the term “platelet-containing fluid” refers to anyfluid, either biological or artificial, which contains platelets.Non-limiting examples of such fluids include various forms of wholeblood, blood plasma, platelet rich plasma, concentrated platelets in anymedium, or the like, derived from human and non-human sources.

As used herein, the term “concentrate” or “concentrating” refers to theseparation of platelets from the bulk of the plasma, whole blood, orother fluid from which it is present. For example, centrifugation,spectrometry, filtration, decanting, gravity settling, or other methodsof concentrating platelets from platelet-containing fluids can be used.When concentrating platelets, it can be desirable to use ananticoagulant (particularly for centrifugation or gravity settling)along with the source of platelets to prevent clotting during theseparation of platelets from other components of the blood, plasma, orother fluid.

The term “anticoagulant” refers to compositions that inhibit clottingwhen concentrating or collecting platelets for use in accordance withexamples of the present disclosure. Anticoagulants generally areavailable as inhibitors of clotting factor synthesis, inhibitors ofthrombin, or antiplatelet drugs. Inhibitors of clotting factor synthesisthat inhibit the production of certain clotting factors in the liver,include compositions such as warfarin (Coumadin). Inhibitors of thrombininterfere with blood clotting by blocking the activity of thrombin, andinclude compositions such as heparin and lepirudin (Refludan).Antiplatelet drugs interact with platelets themselves, and include drugssuch as aspirin, ticlopidine (Ticlid), clopidogrel (Plavix), tirofiban(Aggrastat), eptifibatide (Integrilin), etc.

The terms “lyophilization,” lyophilize,” or the like refer to afreeze-drying or dehydration process that is often used to preserveplatelets, but is used somewhat differently in accordance withembodiments of the present disclosure. Specifically, lyophilization isused primarily not just as a preservative process, but rather, tofurther lyse platelets after initial freeze-thaw or other lysistechnique is conducted. In other words, in accordance with examples ofthe present disclosure, after lysates are formed as described herein,lyophilization provides the added benefit of preserving the growthfactors, cytokines, chemokines, and other contents initially enclosedwithin or bound to the surface the platelets, but which are releasedwhen platelets are lysed as described herein, e.g., freeze-thaw lysing.The process typically works by freezing the material and reducingsurrounding pressure to allow frozen water in the material to sublimatedirectly from the solid phase to the gas phase.

The term “tissue” includes the full range of small tissue sites tocomplete organs.

In accordance with the present disclosure, a “lysate” is the compositionprepared where platelets are destroyed by disrupting their cellmembrane. This can be done chemically, mechanically, by liquidhomogenization, or sonication, but in accordance with certainembodiments described herein, the cytolysis is carried out using afreeze-thaw cycle, and to a lesser degree, as part of the lyophilizationprocess. Freeze-thaw lysates can be formed by freezing a plateletsuspension and then thawing the material to above room temperature,e.g., 30° C. to 45° C., though other freeze-thaw regimens are alsoincluded in the scope of the present disclosure, provided they lead tocytolysis of the platelets. With the freeze-thaw technique, this methodcauses cells to swell and break as ice crystals form, followed bycontraction at thawing. Thus, the cyclical swelling and contractingultimately causes the platelets to break open. Multiple cycles aretypically used for more complete lysis, but the “more complete” lysis isnot necessarily required in accordance with examples of the presentdisclosure. Varying degrees of platelet cytolysis can occur, e.g., atleast 30%, at least 50%, at least 70%, at least 90%, or up to 100%cytolysis, by platelet count.

The term “lyophilized platelet lystates” or “LPL” are prepared asdescribed herein. However, it is noted that the term lyophilizedplatelet lysates also includes “lyophilized platelet rich plasmalysates” or “LPRRL” as a specific type of lyophilized platelet lysates.Thus, any discussion of lyophilized platelet lysates (LPL) also includeslyophilized platelet rich plasma lysates (LPRRL), with the understandingthat one difference is that platelet rich plasma is used or formed aspart of the method or composition. However, in either composition, bothinclude lyophilized platelet lysates.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint. The degree offlexibility of this term can be dictated by the particular variable andwould be within the knowledge of those skilled in the art to determinebased on experience and the associated description herein.

As used herein, a plurality of components may be presented in a commonlist for convenience. However, these lists should be construed as thougheach member of the list is individually identified as a separate andunique member. Thus, no individual member of such list should beconstrued as a de facto equivalent of any other member of the same listsolely based on their presentation in a common group without indicationsto the contrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 0.01 to 2.0” should beinterpreted to include not only the explicitly recited values of about0.01 to about 2.0, but also include individual values and sub-rangeswithin the indicated range. Thus, included in this numerical range areindividual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. This same principleapplies to ranges reciting only one numerical value. Furthermore, suchan interpretation should apply regardless of the breadth of the range orthe characteristics being described.

With these definitions in mind, it has been recognized that thepreparation of lyophilized platelet lysates (LPL) and lyophilizedplatelet rich plasma lysates (LPRPL) can be highly functional for bothresearch and therapeutic applications, either before or afterreconstitution in a liquid carrier. When prepared appropriately, theselyophilized platelet lysates can be stable for extended periods of timewhen frozen, e.g., −80° C., or even at room temperature in certainembodiments. Though these compositions can be useful in both a researchand therapeutic environment without the addition of other ingredients,it is noted that both LPL and LPRPL can include one or more active agentor biologic, such as antifungals, antivirals, antibiotics, growthfactors, or chemicals depending on the research or therapeuticapplication. Furthermore, in certain examples, the LPL or LPRPL can beprepared as part of an encapsulated product, e.g., within liposomes.Liposomes, for example, can be prepared that include multilamellar orunilamellar vesicles. In these examples, LPL or LPRPL can be rehydratedat different concentrations ranging 0.01% to 100%, by volume, and mixedwith a liposome solution within its hydrophobic membrane.

In accordance with this, a method of preparing a composition suitablefor therapeutic use or as a culture medium can comprise steps ofconcentrating platelets from a platelet source to form a platelet richportion of the platelet source, and lysing the platelets in the plateletrich portion to form a plurality of lysates. An additional step canfurther include lyophilizing the lysates to form lyophilized plateletlysates in a composition with released concentrations of availablegrowth factors, cytokines, and chemokines.

In another example, a composition suitable for therapeutic use or as aculture medium can comprise lyophilized platelet lysates prepared fromsource platelets, wherein the lyophilized platelet lysates providereleased concentrations of available growth factor, cytokines, andchemokines. In one example, at least 30% of the source platelets thatremain in the composition, by platelet count, are lysed to form thelyophilized platelet lysates. Other concentrations of lyophilizedplatelet lysates prepared from the source platelets can be at least 50%,70%, 90%, or even up to 100% lyophilized platelet lysates.

In another embodiment, a method of treating mammalian tissue cancomprise applying a composition including lyophilized platelet lysatesto a mammalian tissue site for treatment, wherein the lyophilizedplatelet lysates include released concentrations of available growthfactors, cytokines, and chemokines. In one example, the lyophilizedplatelet lysates are prepared from source platelets, wherein at least30% of the source platelets that remain in the composition, by plateletcount, are lysed to form the lyophilized platelet lysates. Otherconcentrations of lyophilized platelet lysates prepared from the sourceplatelets can be at least 50%, at least 70%, at least 90%, or even up to100% lyophilized platelet lysates.

A method of culturing cells or tissue can comprise admixing a mediacomposition including lyophilized platelet lysates with a cell or tissueculture. The media composition can include lyophilized non-lysedplatelets as well, and at least 30% of a total platelet count can bylyophilized platelet lysates. Other concentrations of lyophilizedplatelet lysates prepared from the source platelets can be at least 50%,at least 70%, at least 90%, or even up to 100% lyophilized plateletlysates.

In each of the various embodiments herein, whether discussing thecompositions or methods, there may be some common features of each ofthese examples that further characterize options in accordance withprinciples discussed herein. Thus, any discussions of the compositionsor methods alone are also applicable to the other embodiment notspecifically mentioned.

In further detail, the present disclosure provides a method forpreparing the lyophilized platelet lysates (LPL), and in one example,LPL can be in the form of lyophilized platelet rich plasma lysates(LPRPL). The platelets can be concentrated using any method known in theart, such as centrifugation or commercially available plateletpurification devices. After platelet or platelet rich plasmapurification or concentration, a resultant concentrated plateletmaterial can be frozen, such as with liquid nitrogen at −190° C. or toat least −80° C. using dry ice, ethanol, or other freeze assistingcomposition, for a period of hours, e.g. 24 hours. After freezing, theconcentrated platelet material can then be quickly thawed to above roomtemperature, e.g., from 30° C. to 45° C., or in one example, at about37° C. This freeze-thaw cycle can be repeated to increase theconcentration of platelet lysates. Generally, the more cycles carriedout, the more platelets that will be lysed.

Once the platelet lysates are formed using the freeze-thaw cyclingprocedure or some other lysing process, LPL (or LPRPL) can then belyophilized using a commercially available lyophilizer, and then storedfor extended periods of times at room temperature or even years at belowfreezing temperatures, e.g., from 0° C. to −100° C., or morespecifically in one embodiment, at about −80° C., the product can bestored for about 5 years or more.

In another aspect of the present disclosure, LPL can be used forresearch applications such as cellular or tissue cultures. Specifically,LPL powder or pellets can reconstituted in a liquid medium (such asDMEM, saline, plasma, DMSO, MEM alpha, RPMI, B-mercaptoethanol,non-essential amino acids, sodium pyruvate, or glutamine, etc.) atappropriate concentrations for a given application. This type of medium,enriched with LPL can be used for culturing mammalian cells,particularly human cells when human platelets are used, for research ortherapeutic applications. It should be noted that the use of LPL isuseful for growing or deriving various types of cells or tissue,including but not limited to, mesenchymal stem cells from varioustissues, human embryonic stem cells, keratinocytes, cardiomyocytes, andmany other cell types within the human body, as these lyophilizedplatelet lysates prepared in accordance with examples of the presentdisclosure contain growth factors and cytokines that allow growth ofcells and tissues, as well as maintain organs and their functions, etc.The growth of cells in LPL supplemented medium, for example, can be animprovement in many cellular systems compared to existing commerciallyavailable medium supplements, such as Fetal Bovine Serum (FBS). Inaddition, LPL cell culture additive can be kept at room temperature forlonger periods of time, which is an advantage over FBS, which expiresafter less than a week at room temperature.

In another aspect of the present disclosure, LPL can be used for manytherapeutic applications, such as a supplement in a medium that will beused to culture cells for therapeutic applications. Thus, LPL can beproduced as described herein, but under a cGMP system, which results inLPL produced under minimally manipulated methods and/or cGMP. Inaddition, it is not required that there be any animal products used toproduce LPL of the present disclosure, making the material highly usefulfor human application.

In another aspect of the present disclosure, LPL can be used to treatwounds, ulcers, or burns. As LPL of the present disclosure can beproduced under cGMP, topical application to damaged human tissue can bebeneficial. As mentioned, LPS can be applied in dry form or rehydratedform prior to application, or as a gel. Alternatively, even if the skinis not damaged by a wound, ulcer, or burn, it can be used for othertypes of damage that occurs as a result of aging, photo damage,pathological or degenerative disease, or the like. Thus, LPL of thepresent disclosure can be used at various concentrations and combinedwith a base cream or other carrier for cosmetic use.

In another aspect, LPL can be rehydrated and nebulized in variousconcentrations and used to treat lung disorders. Fine powders orrehydrated fine droplets can be inhaled into the lungs for treatment oflung disorders, for example. LPL can also be reconstituted in eye dropsas well for treatment of the eyes or surrounding tissue.

In another aspect of the present disclosure, LPL can be rehydrated atany therapeutically effective concentration, or can be kept in powderform, and can be used for orthopedic applications, e.g., bone healing,bone fusion, etc. In yet another aspect, LPL can be combined with abandage that can be applied to the wound directly, or rehydrated thenapplied to the wound. Thus, in part due to the relatively long shelflife at room temperature of LPL of the present disclosure, such abandage has enhanced wound healing properties and will last longer thanmany other compositions, giving it a better chance to remain active andeffective prior to use. Alternatively, other application approaches canalso be used, such as by rehydration and application using a syringe,spray bottle, or other solution dispenser at one of any therapeuticallyeffective concentration when applied to the treatment area. Thus, theadministration of the therapeutic compositions of the present disclosurecan be done in any acceptable manner known in the medical andpharmaceutical arts.

Specific non-limiting examples of administration methods include the useof fluids, aerosols, sprays, mists, lotions, creams, ointments, gels,gums, lozenges, nebulized droplets or powders, suppositories, drops,washes, dispensing bottles, squeeze tubes, pre-soaked fabric, automaticmixing and/or dispensing devices, syringes, bandages, dermal patches orplasters, etc. In one specific example, LPL can be contained in a kitthat can be used in combat or other emergency situations for treatingwounds. The kit can store LPL in a more stable form, and then can bereconstituted for immediate use when needed.

With specific reference to the lysing cycle that occurs, and wherefurther lysing occurs during the lyophilization step as describedherein, increased amounts of growth factors, cytokines, chemokines,etc., can be present in the resultant material. Examples of these growthfactors and other materials that can be present in the resultantmaterial include, without limitation, PDGF, PDAF, VEGF, PDEGF, PF-4,TGF-B, FGF-A, FGF-B, TGF-A, IGF-1, IGF-2, BTG, TSP, vWF, PAI-1, IgG,IgM, IgA, KGF, EGF, FGF, TNF, IL-1, KGF-2, fibropeptide A, fibrinogen,albumin, osteonectin, gro-alpha, vitronectin, fibrin D-dimer, favtor V,antithrombin III, a2 macroglobulin, angiogenim, Fg-D, and elastase. Infurther detail, growth factors, cytokines, or the like that can bepresent and include, without limitation, LIF, anticancer growth factorssuch as IGFBP3, eicosanoids such as PGs orleukotrienes, IL-1 TNF alpha,INFs, TNF-a, IL-6, IL-1(a/b), prostanoid metabolites, complementcomponents, reactive oxygen intermediates, arachidonic acid metabolites,coagulation factors, nitrates, and chemokines. Human derived growthfactors, chemokines, cytokines, and hormones can include alpha defensin,alpha synuclein, beta synuclean, 4-1BBL, 6Ckine, acidic FGF, activin A,avtivin R1b, angiopoietin 2, B-DNF, BAFF, BCA-1, BCA-1, BD-1, BMP-2,BMP-4, BMP-7, BMPRA1, BDNF, CNTF, CTGF, CTLA-4Fc, CXCL1, CXCL2,cardiotrophin-1, Cripto, Cystatin C, Dkk-1, EGF AOF, EGF, EMAP II,ENA-78, EPO, Eotaxin, FGF basic AOF, FGF-10, FGF-16, FGF 17, FGF 18,FGF19, FGF4, FGF6, FGF7, FGF8, FGF8b, FGF9, Flt3, G-CSF, GDNF, GMCSF,HGF, HGH, IFN alpha A, IFN alpha ND, IFN alpha D, IFN alpha a2b, IFN,beta 1A, IFN-gamma, IGF1, IGFII, IGFBP-4, IGFBP6, IL1alpha, IL-1Beta,IL10, IL11, IL12, IL13, IL15, IL17, IL17A. IL17F, IL18, IL19, IL2, IL20,IL21, IL23, IL28A, IL28B, IL29, IL3, IL31, IL33, IL4, IL5, IL6, IL7,IL8, IL9, IL10, ITAC, KGF2, Kallikrein11, Kallikrein4, Kallikrein7,LEFTY-A, LIF, Leptin, MCSF AOF, MCSF, MCP-1, MCP2, MCP3, MCP4, MDC, MIG,MIP1alpha, MIP1 beta, MIP3 alpha, MIP3 beta, MIP4, MIP5, midkine, NAP2,NT3, NT4, Neurotactin, neurturin, Oncostatin, osteoprotegrerin, PDGF-AA,PDGF-AB, PDGF-BB, PTN, Rank ligand, Rank receptor, RANTES<SCF, SCFAOF,SDF-1alpha, SDF-1Beta, CD4, CD40L, TNF-RI, TNFRII, TARC, TECK, TGFalpha, TGF1 Beta1, TGF Beta2, TGF Beta3, TNF beta/lymphotoxin,TNF-alpha, TPO, TRAIL, TWEAK, and VEGF. Thus, the compositions preparedin accordance with examples of the present disclosure can be prepared toremove or destroy the cellular information that is commonly found at thecell membrane, and retain many of these growth factors, cytokines,chemokines, etc., for beneficial use as a healant, cell culture mediumadditive, cosmetic treatment composition, etc.

EXAMPLES

The following examples illustrate embodiments of the present disclosurethat are presently best known. However, it is to be understood that thefollowing are only exemplary or illustrative of the application of theprinciples of the present disclosure. Numerous modifications andalternative compositions, methods, and systems may be devised by thoseskilled in the art without departing from the spirit and scope of thepresent disclosure. The appended claims are intended to cover suchmodifications and arrangements. Thus, while the present disclosure hasbeen described above with particularity, the following examples providefurther detail in connection with what are presently deemed to be themost practical embodiments.

Example 1 Preparation of Lyophilized Platelet Lysates (LPL)

Peripheral blood is collected with an appropriate amount ofanticoagulant to prevent clotting of the blood during processing. Inthis example, 35 mL of blood is collected, which is then centrifuged at200×g (1000 rpm) for 10 minutes at 4° C. with the brake set to off mode.After centrifugation, the platelet rich portion is removed and tubes ofthe platelet rich portion are placed into a freezing range of −190° C.to −80° C. for a period of 24 hours. After 24 hours, the platelets arelysed by thawing quickly at 37° C. After thawing, the tubes are thenreturned for another freeze cycle within the same freezing range for aperiod of 24 hours. The freeze-thaw cycle (one freeze cycle followed byone thaw cycle) is repeated at least once, and more typically, repeatedat least twice for a total 3 cycles, e.g., 3 to 6 freeze-thaw cycles. Itis notable that sonication, filtration, or the like, can be used to lyseor collect platelets as well, either alternatively or additively withrespect to the freeze thaw cycle described herein. After the last thawcycle, the tubes are refrozen at −80° C. and stored overnight. Thefollowing day, the tubes are lyophilized using FreeZone 2.5 Plus(Labconco) under 0.008 mBar at −84° C. for 48 hrs. The lyophilizationcan further lyse additional platelets as well. Depending on the totalvolume to be lyophilized, the time may be increased beyond the 48 hours.Likewise, in some embodiments, the time may be decreased to some degree.The LPL, when prepared in this manner, can be stored at room temperaturefor at least a year, or if at −80° C., for example, a period of 5 yearscan be possible. It is noted that the steps of lysing and lyophilizingcan result in varying degrees of lysed platelets, e.g., 30%, 50%, 70%,90%, or even up to 100% of the platelets can be lysed, depending varyingthe number of freeze-thaw cycles, the parameters of the freeze-thawcycles, the parameters of the lyophilization, etc. Lyophilization alonewill not provide the degree of lysing that can occur when conductingboth steps of lysing and lyophilizing in accordance with embodiments ofthe present disclosure.

Example 2 Preparation of Lyophilized Platelet Rich Plasma Lysates(LPRPL)

The procedure of Example 1 is followed, except for with LPRPL, after theblood is collected as previously described, it is processed using acommercially available PRP preparation device such as the Magellan PRPdevice by Arteriocyte Medical Systems. The lysing and lyophilizationsteps of Example 1 can be otherwise the same for the present Example.

Example 3 Use of LPL or LPRPL for Culturing Human Mammalian Cells

After preparing the LPL of Example 1 or the LPRPL of Example 2, thematerial can be used to supplement tissue culture media for human cellculture. For example, a concentration of LPL or LPRPL can range from0.5% to 20% by volume of total lyophilized LPL or LPRPL used for thecell culture. In one specific example, 10 mL of LPL or LPRPL can be usedto prepare 100 mL of media.

In one specific embodiment, cultured human mesenchymal stem cells areisolated from bone marrow and adipose tissue, and a media compositioncan be prepared as follows:

10 mL lyophilized LPL or LPRPL (about 1 gram)

1 mL of NEAA (100×)

1 mL of Glutamax (100×)

1 mL of Penn/Strep (100×)

97 mL of DMEM LG—without Phenol

In this example, the cells are cultured until about 80% confluency andpassaged to generate the desired cell number.

Example 4 Use of LPL or LPRPL for Treating Wounds (Rehydrated Method)

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL can then be rehydrated in a syringe or a spray bottle at adesired predetermined therapeutic concentration, such as 5%, 10%, 20%,30%, 40%, 50%, etc., by volume). The concentration can be related to adesired effect, stability, need for specific type of wound to betreated, etc. In any event, the rehydrated LPL or LPRPL is then applieddirectly onto the site to be treated, and is covered. The applicationcan be a part of one time treatment, or more typically, 25E1 can berepeated daily, or every three to five days as desired.

Example 5 Use of LPL or LPRPL for Treating Wounds (Non-Rehydrated orPowder Method)

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL is ground into a fine powder and applied directly onto a woundor site of treatment without first rehydrating the composition. Thewound is covered for treatment at the wound or treatment site. Theapplication can be a part of one time treatment, or more typically, canbe repeated daily, or every three to five days as desired.

Example 6 Use of LPL or LPRPL for Treating Wounds Using a Bandage

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL is ground into a fine powder and placed into a small porousgauze. The gauze is then attached to an adhesive material to form abandage. In this condition, the LPL or LPRPL bandage is ready to beapplied onto various types of wounds directly. Alternatively, the LPL orLPRPL bandage can be rehydrated with saline or water and then placedonto the wound site.

Example 7 Use of LPL or LPRPL for Treating Lung Disorders

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL is ground into a fine powder and then can be rehydrated atvarious concentrations determined to have a therapeutic effect for agiven treatment regimen. In this example, a concentration of 10% byvolume was used. For direct treatment to the lungs, the rehydrated LPLor LPRPL is nebulized and inhaled by the patient for a period of 10minutes.

Example 8 Two-Part Rehydration of LPL or LPRPL

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL is ground into a fine powder and placed into one side of amulti-compartment vessel, and on the other side of the vessel, a sterilesaline is placed so that the two materials do not contact one anotheruntil a seal is broken. Once the seal is broken, the saline mixes withthe LPL or LPRPL and they become rehydrated. A spray applicator is thenturned to the ON position, and using a pump to press up and down, thereconstituted LPL or LPRPL is applied by spraying directly onto a siteto be treated. This is one example of a device that can be used inaccordance with examples of the present disclosure. Other devices canalso be used for reconstitution and/or application of the LPL or LPRPLdescribed herein.

Example 9 Use of LPL or LPRPL with an Active Agent or Biologic

After preparing the LPL of Example 1 or the LPRPL of Example 2, the LPLor LPRPL is ground into a fine powder or can be rehydrated at variousconcentrations determined to have a therapeutic effect for a giventreatment regimen. The LPL or LPRPL is then admixed with an active agentor biologic, such as an antifungal, antiviral, antibiotic, growthfactor, or chemical, depending on the therapeutic or researchapplication. The concentration of active agent or biologic can be anyconcentration that has a therapeutic effect or meets a research goalthat is being considered, without limitation.

Example 10 Comparison of Mesenchymal Stem Cell Cultures Using VariousMedia

Lyophilized platelet lysates prepared similarly to that described inExample 1 were compared to other media products for culturingmesenchymal stem cells. FIG. 1 graphically shows the results of thestudy. Specifically, Media 1 included Embryonic Stem Cell (ESC)qualified 10% Fetal Bovine Serum (FBS); Media 2 included 10% PlateletRich Plasma (PRP); Media 3 included 10% Platelet Lysates (PL); and Media4 included 5% lyophilized platelet lysates (LPL), each percentage byvolume. As can be seen from FIG. 1, the cells cultured in the mediaincluding the lyophilized platelet lysates significantly outperformedthe cell cultures that did not include lyophilized platelet lysates.

While the invention has been described with reference to certainpreferred embodiments, those skilled in the art will appreciate thatvarious modifications, changes, omissions, and substitutions can be madewithout departing from the spirit of the invention. It is thereforeintended that the invention be limited only by the scope of the appendedclaims.

What is claimed is:
 1. A method of treating mammalian tissue, comprisingapplying a composition including lyophilized platelet lysates to amammalian tissue site for treatment, wherein the lyophilized plateletlysates include released concentrations of available growth factors,cytokines, and chemokines.
 2. The method of claim 1, wherein thelyophilized platelet lysates are prepared from source platelets, andwherein at least 30% of the source platelets that remain in thecomposition, by platelet count, are lysed to form the lyophilizedplatelet lysates.
 3. The method of claim 1, wherein the lyophilizedplatelet lysates are from human platelets, and the mammalian tissue siteis a human tissue site.
 4. The method of claim 3, wherein the tissuesite is a skin site.
 5. The method of claim 3, wherein the treatment iscosmetic.
 6. The method of claim 3, wherein the treatment is as a woundhealant.
 7. The method of claim 1, wherein the tissue site is aninternal tissue site.
 8. The method of claim 7, wherein the internaltissue site is lung tissue.
 9. The method of claim 7, wherein theinternal tissue site is exposed as a result of surgery.
 10. The methodof claim 1, wherein the lyophilized platelet lysates are applied in theform of a powder.
 11. The method of claim 1, wherein the lyophilizedplatelet lysates are applied in the form of a reconstituted lysatesuspension.
 12. The method of claim 11, wherein the reconstituted lysatesuspension comprises the lyophilized platelet lysates brought togetherwith saline for application to the tissue site.
 13. The method of claim11, wherein the tissue site is the eye.
 14. The method of claim 1,wherein the lyophilized platelet lysates are applied in the form of anebulized inhalant.
 15. The method of claim 1, wherein the lyophilizedplatelet lysates are applied in the form of an active ingredient on abandage.
 16. The method of claim 1, wherein the lyophilized plateletlysates are in the form of lyophilized platelet rich plasma lysates. 17.The method of claim 1, wherein an active agent or biologic is applied tothe tissue site along with the lyophilized platelet lysates.
 18. Themethod of claim 1, wherein the lyophilized platelet lysates are preparedfrom source platelets, and wherein at least 50% of the source plateletsthat remain in the composition, by platelet count, are lysed to form thelyophilized platelet lysates.
 19. The method of claim 1, wherein thelyophilized platelet lysates are prepared from source platelets, andwherein at least 70% of the source platelets that remain in thecomposition, by platelet count, are lysed to form the lyophilizedplatelet lysates.
 20. The method of claim 1, wherein the lyophilizedplatelet lysates are prepared from source platelets, and wherein atleast 90% of the source platelets that remain in the composition, byplatelet count, are lysed to form the lyophilized platelet lysates.